The invention concerns a bilirubin oxidase (E.C.1.3.3.5) from plants, in particular from the genus Alfalfa, a process for the isolation as well as the use of the enzyme for the determination of bilirubin or the removal of bilirubin in aqueous liquids, in particular biological liquids.
A number of enzymes with bilirubin oxidase activity from various plant sources are known. Such bilirubin-specific enzymes are isolated in particular from strains of the genus Bacillus (U.S. Pat. No. 4,770,997), Myrothecium and Coprinus (DE 32 39 236), Solanaceae, Musaceae and Liliaceae (EP 0 140 004), Compositae (EP 0 247 846) and from fungi (EP 0 005 637) and citrus fruits (EP 0 320 095). However, the bilirubin-specific enzymes isolated from the aforementioned plant genera characteristically have a relatively low specificity for bilirubin and have various side activities. The latter are a particular hindrance in diagnostic analytical methods in which a number of other components, some of which are similar to bilirubin, can be present as a result of individual differences in the composition of the respective biological sample such as blood, urine or serum. This can in addition be caused by the degradation of certain pharmaceutical agents. In the case of most known enzymes with bilirubin oxidase activity such interferences occur in particular when phenolic compounds such as catechol, hydroquinone or phenol itself are present. This non-specificity of the enzymes, i.e. an undesired phenol oxidase activity, leads to delayed reactions in the corresponding determinations and the enzymes are therefore not suitable for an application in colorimetric tests or only to a limited extent.
Other known bilirubin oxidases have a low specific activity or a high Michaelis constant for bilirubin so that interferences can occur due to the large amount of protein or enzyme which has to be added to the test in such cases.
In addition the stability at higher temperatures is not adequate in the case of a number of the previously available bilirubin oxidases. In the corresponding determinations this causes a non-linear time course of the kinetics.
The objects of the invention are therefore to provide an enzyme with bilirubin oxidase activity which has a high specificity for bilirubin and does not have any interfering contaminating activities and at the same time posesses a high thermostability and can be isolated in adequate amounts from a readily available source of raw material.